Modulation of the E-cadherin in human cells infected in vitro with Coxiella burnetii

High concentration of soluble E-cadherin (E-cad) was previously found in sera from Q fever patients. Here, BeWo cells which express a high concentration of E-cad were used as an in vitro model to investigate the expression and function of E-cad in response to infection by Coxiella burnetii, the etiological agent of Q fever. Infection of BeWo cells with C. burnetii leads to a decrease in the number of BeWo cells expressing E-cad at their membrane. A shedding of soluble E-cad was associated with the post-infection decrease of membrane-bound E-cad. The modulation of E-cad expression requires bacterial viability and was not found with heat-inactivated C. burnetii. Moreover, the intracytoplasmic cell concentration of β-catenin (β-cat), a ligand of E-cad, was reduced after bacterial infection, suggesting that the bacterium induces modulation of the E-cad/β-cat signaling pathway and CDH1 and CTNNB1 genes transcription. Finally, several genes operating the canonical Wnt-Frizzled/β-cat pathway were overexpressed in cells infected with C. burnetii. This was particularly evident with the highly virulent strain of C. burnetii, Guiana. Our data demonstrate that infection of BeWo cells by live C. burnetii modulates the E-cad/β-cat signaling pathway.


Introduction
Coxiella burnetii, a known intracellular bacterium causing Query (Q) fever in humans, is most often transmitted to humans through products derived from infected animals [1], and primarily targets the lungs. The bacterium infects a large spectrum of susceptible cells, including myeloid cells [2,3], trophoblasts [4] and adipocytes [5]. Human primary infection remains asymptomatic in most people (around 60% of cases) [6]. In the other cases, symptoms develop within 2-6 weeks after C. burnetii exposure and mainly consist of hepatitis, endocarditis, pneumonia, vascular infection and lymphadenitis [7,8]. Although these symptoms usually resolve in a few weeks, in rare cases (less than 5%), the infection become persistent and progresses to chronic endocarditis [9]. Factors that may determine the severity of the disease remain mostly unknown [10][11][12][13], but considerable genomic heterogeneity was reported among C. burnetii strains [14][15][16][17][18], and gene deletion was associated with a higher strain virulence [19][20][21]. A role for the bacterial lipopolysaccharide (LPS) was also highlighted, since several in vitro passages a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 of the virulent Nine Mile strain of C. burnetii generate an avirulent strain characterized by a truncated LPS with the absence of the terminal sugar-containing O-polysaccharide chain [22,23].
In the past decade, a diagnosis of Q fever with persistent focal infection was reported as an increased risk factor for B-cell lymphoma [24][25][26]. Overproduction of interleukin (IL)-10, a Bcell growth factor [27], is critical for sustaining C. burnetii replication [28], and might possibly favor lymphoma occurrence [25]. In peripheral blood mononuclear cells (PBMCs) of Q fever patients it was possible to characterize a transcriptional signature (overexpression of genes involved in the anti-apoptotic process or repression of pro-apoptotic pathways), that could be, in very rare cases, associated with the development of non-Hodgkin lymphoma (NHL) [29]. We also speculated that if C. burnetii infection could sometimes promote the development of NHL, the early steps towards NHL progression might be the decrease of E-cadherin (E-cad) surface expression on the CD20 + B-cells subpopulation of PBMCs (E-cad positive B-cells represents less than 1% of the circulating B lymphocytes) from C. burnetii-infected patients [30]. However, until now the molecular mechanisms that could account for the progression of the disease to lymphoma have remained elusive. Moreover, these results are controversial, and other studies of Q fever patients have not confirmed the link between Q fever and NHL [31,32]. A recent large analysis of the Dutch population incorporating the 2007-2010 Q fever outbreak (266 million people including 61424 diagnosed with NHL and 4310 persons diagnosed with acute Q fever), concluded to the lack of increased risk of NHL after Q fever [33].
We previously found that soluble E-cad (sE-cad) is present in higher concentrations in sera of Q fever patients than healthy controls [30], and postulated that E-cad cleavage may be a step towards C. burnetii-invasion of the host during Q fever. Increasing evidence can be found in the literature indicating that cadherins may play a major role during the development of infectious diseases [34]. Among cadherins, the E-cadherin (E-cad), a 120 kDa adhesion cell-surface protein, is known for its role in cell-to-cell tight junctions ensuring integrity of epithelial barriers. E-cad also behaves as a signaling molecule through its intra-cytoplasmic tail that binds second messengers, thereby having roles in cell activation, cell division, and cell differentiation and/or invasion [35,36]. Besides its role in inter-cellular adhesion, E-cad behaves as a tumor suppressor [37][38][39] and plays a major role in cancer invasion/metastasis [40][41][42]. Cell reprogramming can be achieved by the release of sE-cad generated from the cleavage of the extracellular domain of E-cad [43]. E-cad is known to be a target for several bacterial sheddases, leading to the release of sE-cad [43,44].
Here we developed an in vitro cellular model with BeWo cells which express a high concentration of E-cad at their surface [45] and are susceptible to C. burnetii infection [4] to study the effect of C. burnetii infection on the expression of E-cad and evidenced that C. burnetii infection modulates expression of the E-cad/β-catenin signaling pathway by acting on CDH1 and CTNNB1 gene transcription, on cell-surface expression of E-cad, the release of sE-cad and the cytosolic pool of β-catenin.

Coxiella burnetii
Coxiella burnetii (Nine Mile strain RSA496, Guiana Strain Cb175) was grown as described [46]. L929 cells line were used for in vitro culture using a Minimum Eagle medium (MEM, Invitrogen, USA) supplemented with 4% fetal bovine serum (FBS, Invitrogen, USA) and 1% 2mM L-glutamine (Invitrogen, USA) and then incubated at 35˚C in a 5% CO 2 atmosphere. After three passages infected cells were sonicated, and the cell-free supernatants were centrifuged to harvest the bacteria, which were then washed and stored at -80˚C until use. Gimenez staining and qPCR (using the com-1 gene) were used to determine the concentration of bacteria in the sample.

In vitro cell culture model
Blood samples (leucopacks) used in our study come from the French Blood Establishment (Etablissement français du sang, EFS), which carries out donor inclusions, informed consent and sample collection. Through a convention established between our laboratory and the EFS (No.7828), buffy coats were obtained from healthy blood donors. Peripheral blood mononuclear cells (PBMCs) were isolated after centrifugation on Ficoll cushions (MSL, Eurobio, France). Monocytes (5-15% of the total number of PBMCs) were obtained from PBMCs using magnetic beads coated with monoclonal Abs directed against CD14, according to the manufacturer's instructions (Miltenyi Biotech, France). Monocytes-derived-macrophages (MDM) were obtained following the incubation of monocytes with 10% heat-inactivated human AB serum (MP Biomedicals, LLC, France) during a 3-day culture followed by 4 days of incubation with 10% FBS (Invitrogen, USA) as previously described [47]. Monocyte-derived-dendritic cells (moDC) were obtained by incubation of monocytes with 1 ng/mL of granulocytes-macrophages colony-stimulating factor (GM-CSF, R&D Systems, France) and Interleukin-4 (R&D Systems, USA) for 7 days as previously described [48]. Mast cells were obtained from PBMCs as previously described [49]. All cells were cultured in Roswell Park memorial institute (RPMI) 1640 medium (Gibco, Thermo Fisher, USA) supplemented with 10% FBS.
The human trophoblastic BeWo cell line was purchased from the American type culture collection (ATCC, CCL-98, Bethesda, USA) and cultured in a Dulbecco's Modified Eagle Medium F-12 Nutrient Mixture (DMEM F-12, Invitrogen, USA) containing 10% FBS. The HeLa cell line (ATCC CCL-2), a human epithelial cell line derived from a cervical cancer, was cultured in DMEM supplemented with 10% FBS and 1% L-glutamine (Invitrogen, USA). MRC-5 (ATCC CCL-171), a human embryonic fibroblast cell line obtained from an aborted fetus, was cultured in MEM supplemented with 4% FBS and 1% L-glutamine. THP-1 (ATCC TIB-202), a human monocytic cell line derived from an acute monocytic leukemia patient, was maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) at 37˚C and 5% CO 2 .

Microarray and data analysis
The microarray was performed as previously described [29] with data submitted to NCBI's Gene Expression Omnibus (GEO series accession number GSE112086). Briefly, RNAs were extracted using an RNeasy Mini Kit (QIAGEN SA, France) with a DNase I step, then labeled using Cyanin-3 CTP (Agilent Technologies) and hybridized on chips containing 45000 probes (4x44K Whole Human Genome). After hybridization, slides were washed and scanned with a pixel size of 5μm using the DNA Microarray scanner G2505C. The raw data were extracted (Extraction Software 10.5.1) and processed using GeneSpring GX 14.9 software (Agilent Technologies). A selection filter of both statistical value p<0.01 (t-Student test) and an absolute value of fold change (FC) greater than 1.5 was used to identify differentially expressed genes. Modulated genes were analyzed using ClustVis software. ratio, or stimulated with 100 ng/mL of lipopolysaccharide (LPS) from E. Coli (O55:B5; Sigma-Aldrich, USA) for 48 h at 37˚C in a 5% CO 2 atmosphere. RNAs were extracted as described above. The first-strand cDNA was obtained using oligo(dT) primers and Moloney murine leukemia virus-reverse transcriptase (MMLV-RT kit; Life Technologies, USA), using 100 ng of purified RNA. The qPCR experiments were performed using specific oligonucleotide primers and hot-start polymerase (SYBR Green Fast Master Mix; Roche Diagnostics, Germany). The amplification cycles were performed using a C1000 Touch Thermal cycler (Biorad, USA). Specific primers used in this study are listed in Table 1 and the results were normalized using the housekeeping gene β-actin (ACTB) (Fwd: 5' CAT GCC ATC CTG CGT CTG GA 3'; Rev: 5' CCG TGG CCA TCT CTT GCT CG 3') and expressed as the relative expression (2 -ΔCT ) where ΔCt = Ct Target − Ct Actin , and as the fold change (2 -ΔΔCT ), where ΔΔCt = ΔCt stimulated -ΔCt unstimulated as previously described [50]. Ct values were defined as the number of cycles for which fluorescence signals were detected.

E-cadherin protein quantification
BeWo cells (5x10 5 cells/well) were cultured in flat-bottom 12-well plates for 12 h and were then infected with live C. burnetii NMI strains or exposed to heat-inactivated C. burnetii strains at a 50:1 bacterium-to-cell ratio or stimulated with 100 ng/mL of LPS for 4, 24 and 48 h at 37˚C in a 5% CO 2 atmosphere.
For each kinetics, the culture supernatants were collected, centrifuged at 1000 g for 10 min and stored at -20˚C until use. The quantity of sE-cad in the supernatants was determined using a specific immunoassay (DCADEO, R&D Systems, USA) according to the manufacturer's instructions. The minimal detectable concentration of human sE-cad was 0.313 ng/mL. Protein quantification was evaluated using a western blot assay. Cells were washed with ice cold phosphate buffered saline (PBS 1X) and lysed using 1X RIPA buffer (100 mM Tris-HCl pH 7.5, 750 mM NaCl, 5 mM EDTA, 5% IGEPAL, 0.5% sodium dodecyl sulfate (SDS), and 2.5% Na deoxycholate) supplemented with both a protease and phosphatase cocktail inhibitor (Roche, Germany). Ten μg of protein were loaded onto 10% SDS polyacrylamide gels. After transfer into a nitrocellulose membrane, the blots were incubated overnight at 4˚C with a saturation solution (5% fat free milk (FFM)-1X PBS-0.3% Tween 20). Blots were then incubated with mouse anti-human E-cad ectodomain mAb (1:5000) (HECD-131700, Invitrogen, USA) for 2 h, followed by an incubation with mouse anti-human E-cad cytoplasmic domain mAb (4A2C7, Life Technologies). In some experiments, β-catenin was detected using an anti- Table 1. Specific primers used for q-RTPCR assays.

Genes (symbols) Primers sequence
Sense Antisense human β-catenin antibody (1:1000) (Agilent Dako, USA). After three washes in 1X PBS-0.3% Tween 20, the blots were incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:10000) for 2 h at room temperature. β-actin expression was measured using an antihuman β-actin horseradish peroxidase-conjugated mAb (1:25000, Life Technologies, USA) as the loading control. The proteins were revealed using an ECL western blotting substrate (Promega, USA) and images were digitized using a Fusion FX (Vilber Lourmat, France). The density of the bands was measured using Image J v 1.8.0.

Confocal immunofluorescence assay
BeWo cells were cultured on sterile coverslips in 24-well plates at an initial concentration of 2×10 5 cells/well and were then infected with live virulent C. burnetii NMI strains or exposed to heat-inactivated virulent C. burnetii NMI strain at a 50:1 bacterium-to-cell ratio or stimulated with 100 ng/mL LPS for 24 h at 37˚C in a 5% CO 2 atmosphere. After fixation with 4% paraformaldehyde, cells were permeabilized with 0.1% Triton X-100 for 3 min and saturated with 3% BSA-0.1% Tween 20-PBS for 30 min at room temperature. Fixed cells were first incubated with a mouse anti-E-cad cytoplasmic domain mAb (1:1000) (4A2C7, Life Technologies, USA) for 1 h at room temperature and then with 1:500 rabbit polyclonal anti-C. burnetii antiserum. After washing, cells were incubated for 30 min at room temperature with a mix (1:1000) of goat anti-rabbit IgG secondary antibody (Alexa Fluor 647, Life Technologies, USA) and goat anti-mouse IgG secondary antibody (Alexa Fluor-555, Life Technologies, USA). 4',6'-diamino-2-fenil-indol (1:25000) (DAPI, Life Technologies, USA) and Phalloidin (1:500) (Alexa-488, OZYME, France) were used for staining the nucleus and the filamentous actin, respectively. Labelled cells were analyzed using laser scanning confocal microscopy and pictures were acquired using a confocal microscope (Zeiss LSM 800) with a 63X/1.4 oil objective, an electronic magnification of 0.7 and a resolution of 1014_1014 pixels.

Flow cytometry assay
BeWo cells (1×10 6 cells/well) were cultured in flat-bottom 6-well plates and were then infected with a live virulent C. burnetii NMI strain at a 50:1 bacterium-to-cell ratio or stimulated with 100 ng/mL LPS for 24, 48 and 72 h at 37˚C in a 5% CO 2 atmosphere. Cells were incubated with 2 mM EDTA in PBS for 15 min at 4˚C to release the adherent cells from the culture plate. After centrifugation at 500 g for 5 min, the pellet was suspended in a FACS buffer (2 mM EDTA, 10% FBS in PBS) and then incubated for 30 min with a mouse anti-human E-cad ectodomain antibody (HECD-131700, 1:1000). After two washes, cells were incubated with a goat anti-mouse IgG Alexa Fluor-555 secondary antibody. Fluorescence intensity was measured using a Canto II cytofluorometer (Becton Dickinson/Biosciences, France) and the results were analyzed using FlowJo V10.7.2 software (Becton Dickinson, USA).

Electron microscopy analysis
BeWo cells (1×10 6 cells/well) were cultured in flat-bottom 6-well plates and were then infected with live virulent C. burnetii NMI and GuiI strains at a 100:1 bacterium-to-cell ratio at 37˚C in a 5% CO 2 atmosphere. After 24 h of infection, the cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for five hours at 4˚C. Resin embedding was microwave-assisted with a Biowave Pro+ (Pelco, Fresno, CA, USA). After washing twice with a mixture of 0.2-M saccharose/0.1-M sodium cacodylate and once with distilled water, samples were progressively dehydrated by successive baths in 50%, 70% and 96% ethanol. Substitution with LR-White resin (medium grade; Polysciences, Warrington, PA, USA) was achieved by incubations with 25% to 100% LR-White resin and samples were placed in a polymerization chamber for 72 h at 60˚C. All solutions used above were 0.2-μm filtered. After curing, the resin blocks were manually trimmed with a razor blade and dish bottoms were detached from cell monolayers by cold shock via immersion in liquid nitrogen for 20 seconds. Resin blocks were placed in a UC7 ultramicrotome (Leica), trimmed to pyramids, and ultrathin 100 nm sections were cut and placed on HR25 300 Mesh Copper/Rhodium grids (TAAB, Aldermaston, England). Sections were contrasted with uranyl acetate and lead citrate. Grids were attached with double sided tape to a glass slide and platinum-coated at 10 mA for 20 seconds with a MC1000 sputter coater (Hitachi High-Technologies, Japan). Electron micrographs were obtained on a SU5000 SEM (Hitachi High-Technologies, Japan) operated in high-vacuum at 10 kV accelerating voltage and observation mode (spot size 30) with BSE detector.

Statistical analysis
The statistical analyses of the data were performed using GraphPad-Prism software (version 6.0). The results are presented as the ± standard error of the mean (SEM). The Mann-Whitney U test was used for flow cytometry, the two-way ANOVA test for E-cadherin quantification and transcriptional analysis, and a nonparametric Kruskal-Wallis with Dunn's multiple comparison test for group comparison. A p value <0.05 was considered statistically significant.

Selection of the BeWo cell line as an appropriate cellular model to study Ecad expression in cells infected with C. burnetii
It was previously reported that many cell types, including myeloid cells [51,52], trophoblasts [4,53], lung cells [54,55], and adipocytes [5] are susceptible to C. burnetii infection. To conduct our study, it was first necessary to select a cellular model with high expression of E-cad. To this end, we evaluated E-cad expression in different cell lines, including HeLa (human cervical cancer cell line), THP-1 (human monocytic cell line), MRC-5 (human fetal lung fibroblasts), BeWo (human placenta choriocarcinoma cell line), and primary monocytes from healthy donors. As shown in Fig 1A, using a western blot assay, BeWo cells were found to express the highest concentration of E-cad and HeLa cells ranked second. Additionally, we also confirmed the highest membrane expression of E-cad on BeWo cells by confocal microscopy (Fig 1B). This finding was consistent with the reports from the literature on the common use of BeWo cells as a model to study E-cad expression [56,57]. Moreover, scanning electron microscopy of C. burnetii-infected BeWo cells (Figs 1C and S1) showed the presence of several vacuoles, either empty or containing electron-dense circular structures, with diameters ranging from 250 nm to 700 nm dispersed in the cell cytoplasm, corresponding to the morphology and size of C. burnetii as previously described [58]. Therefore, even if other tested cells are susceptible to infection by the bacterium, the BeWo cell line was chosen for further study due to the fact that it exhibits both susceptibility to C. burnetii infection and high expression of E-cad, which represents optimal experimental conditions for monitoring of the release of the soluble fraction of the protein.

C. burnetii infection modulates cell surface expression of E-cad
The level of E-cad protein expression at the cell surface of BeWo cells infected or not by C. burnetii NMI was evaluated by flow cytometry, targeting the ectodomain portion of E-cad. Twenty-four hours post-C. burnetii infection, the percentage of BeWo cells expressing membrane-bound E-cad decreased significantly compared to LPS stimulated-and unstimulatedcells (p<0.05 and p<0.01, respectively) used as controls (Fig 2A and 2B). Also, we found a

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E-cadherin pathway modulation during Coxiella burnetii infection significant decrease in the mean intensity fluorescence signal of the E-cad in BeWo cells infected with C. burnetii compared to LPS-stimulated and unstimulated cells (p<0.05 and p<0.01, respectively) (Fig 2C). Then, we studied whether bacterial antigen/cell contact was sufficient to trigger E-cad modulation, or whether infection was absolutely required to observe a change in E-cad expression. Thus, we investigated by confocal microscopy E-cad expression on BeWo cells after 4 hours and 24 hours of infection with live C. burnetii, while controls consisted of exposure to heat-inactivated bacteria or LPS. Although for unstimulated BeWo cells a high level of fluorescence and homogeneity of E-cad distribution was observed, this result was not limited to this experimental condition. The same expression profiles were observed for BeWo cells after E. coli LPS stimulation or heat-inactivated C. burnetii bacterium stimulation (Fig 2D). Notably, a short-term intracellular colocalization of the E-cad and C. burnetii fluorescence signals was found in BeWo cells at early stage of infection with live C. burnetii (

Release of sE-cad by C. burnetii-infected BeWo cells and modulation of CDH1/E-cad and CTNNB1/β-catenin gene transcription
Since we previously reported that Q fever patients have high expression of serum sE-cad [30], we aimed at investigating whether the decreased expression of membrane-bound E-cad in C. burnetii-infected BeWo cells could be associated with a release of sE-cad after E-cad proteolysis. As shown in Fig 3A, we measured progressive basal accumulation of sE-cad in the cell culture medium over time (at 24 hours and 48 hours). Interestingly, sE-cad release was significantly higher (p <0.001) over time in infection conditions compared to control conditions (unstimulated and LPS-stimulated BeWo cells). A further immunoblot detection of the products synthesized in C. burnetii-infected cells evidenced two components: the integral 120 kDa E-cad, and a protein of about 60 kDa, a proteolytic product of E-cad (Fig 3C). In contrast, only the 120 kDa E-cad was found in the control experiments. It is worth noting that the expression of β-catenin, an intracellular ligand of E-cad, was strongly decreased in BeWo cells under live C. burnetii-infection conditions. This was further confirmed using a densitometry scanning of the band that showed a significant (p<0.05) decrease of β-catenin in C. burnetiiinfected BeWo cells.
In accord with the literature regarding the E-cad/β-catenin signaling pathway, our results suggest a translocation of β-catenin into the cell nucleus, associated with a modulation of CDH1/E-cad gene expression. To confirm this hypothesis, the expression of CDH1/E-cad and CTNNB1/β-catenin gene transcription was analyzed by qRT-PCR. Both CDH1 and CTNNB1 genes were found significantly overexpressed (p<0.01 and p<0.05, respectively) in BeWo cells with C. burnetii infection compared to cells exposed to E. coli-LPS (Fig 3B).
These results indicate that C. burnetii infection of BeWo cells induces a shedding of the Ecad protein ectodomain at an early stage of the infection (detectable at 24 hours post-infection) and suggest that sE-cad shedding is associated with the nuclear translocation of β-catenin and subsequent up-regulation of CDH1 and CTNNB1 gene transcription through a feedback control loop.

C. burnetii modulates expression of genes from both the E-cad/β-catenin pathway and the Wnt/frizzled/β-catenin pathway in BeWo cells
To further demonstrate that the overexpression of CDH1 and CTNNB1 genes found in C. burnetii infection of BeWo cells is not strain-dependent, BeWo cells were exposed for 48 hours to either the live Nine Mile strain of C. burnetii or the more virulent Guiana (Cb175) laboratory strains of C. burnetii. Heat-inactivated C. burnetii (Nine Mile strain) and E. coli LPS were used as controls. As shown in Fig 4A and 4B, a significant overexpression of CDH1 and CTNNB1 genes was found in BeWo cells infected with each of the two live strains of C. burnetii, extending our earlier observations, illustrated in Fig 3C. In contrast, when BeWo cells were exposed to heat-inactivated bacteria, CTNNB1 gene expression was further increased and CDH1 was significantly decreased. These data suggest that the interaction of bacterial antigens with BeWo cells is sufficient to trigger signals leading to CTNNB1 gene transcription, while induction of CDH1 requires infection.
We then explored the expression of several other genes known to encode proteins linked to the E-cad/β-catenin and Wnt/frizzled/β-catenin axis, including WNT3A, WNT5A, WNT9b, FZD1, APC, NKD2, BCL9, TCF7L2 and CNND1. As shown in Fig 4C, most of these genes were overexpressed in C. burnetii-infected BeWo cells, some of which (e.g., BCL9, Wnt3A) being also overexpressed when BeWo cells were exposed to heat-inactivated-bacteria infected cells. Notably, BCL9, WntA3, and FDZ1 genes were significantly overexpressed where cells were infected with the live and more virulent Guiana strain of C. burnetii compared to infection with the live Nine Mile strain. We also observed that the gene coding for the tumor suppressor APC was significantly overexpressed after BeWo cell exposure to E. coli LPS. It is worth noting that the CCND1 gene coding for cyclin D1 kinase, a cell cycle activator and oncogenic protein, was significantly over-transcribed in cells infected with live C. burnetii, notably in cells infected with the Nine Mile strain. The CCND1 gene is much less expressed with heatinactivated bacteria, suggesting that activation of this gene requires infection of cells by live bacteria. In addition, we also used a micro-array technology to compare the expression of a set of 45000 genes in BeWo cells, infected or not by C. burnetii. This approach indicated that 12189 genes were down-modulated after live C. burnetii infection of BeWo cells, while 6492 genes were found up-regulated, among which genes such as CDH1, CCND1, and FZD1 were found highly expressed compared to their transcription levels in uninfected BeWo cells (S4 Fig).
The transcriptomic results illustrated in Fig 4 were subjected to a principal component analysis (PCA) and a hierarchical clustering heatmap was regenerated. This analysis highlighted distinct gene expression profiles (Fig 5), the first one corresponding to live C. burnetii strain infectious conditions, with slight differences between live C. burnetii strains, while other profiles corresponded to unstimulated cells, LPS stimulated cells and cells exposed to heat-inactivated C. burnetii. Genes activated by the live C. burnetii strain infection cluster together and show a mirrored gene expression compared to other conditions. These results indicate that live C. burnetii specifically induce CDH1, CCND1, CTNNB1 and BCL9 gene overexpression in BeWo cells. However, for induction of the BCL9 gene, infection is not required, and transcription can be stimulated by simply exposing the cells to C. burnetii bacterial antigens.

Discussion
We report here the first experimental evidence that differential gene expression analysis shows modulation of the E-cadherin/β-catenin pathway signature during C. burnetii infection in vitro and that this modulation results in disruption of E-cad expression on the cell surface. As

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E-cadherin pathway modulation during Coxiella burnetii infection a strictly intracellular bacterium, and in order to establish a replication niche, C. burnetii forms intimate interactions with its hosts, which aid in avoidance of the host immune response. In addition to the immune response, C. burnetii must overcome another central host defense mechanism, apoptosis. The molecular crosstalk between the bacterium and the host affects the induction of apoptosis, thereby prolonging its survival.
We previously reported that activation of protein tyrosine kinases (PTK) by C. burnetii in TPH-1 monocytes reflects C. burnetii virulence, since avirulent variants were unable to stimulate PTK [59]. Additionally, modulation of cell signaling following in vitro C. burnetii infection of the TPH-1 human macrophage-like cell line was also reported by Voth and Heinzen [60], who found that infection directs the sustained activation of host pro-survival kinases Akt and Erk1/2, necessary for anti-apoptotic activity that can conduct the development of certain lymphomas. Menelotte et al. [25] were able to link the development of B-cell non-Hodgkin lymphoma to C. burnetii infection. Despite intensive work to identify the bacterial compounds supporting C. burnetii replication and virulence [11][12][13], the molecular mechanisms involved in C. burnetii-induced Q fever and possibly associated NHL remain largely unknown. In more recent investigations [29] and working on Q fever patient samples, we found that specific genes involved in anti-apoptotic processes (e.g., BCL-2) were highly expressed, whereas proapoptotic genes were repressed in PBMCs from patients with C. burnetii-associated NHL, supporting possible involvement of the corresponding proteins in lymphomagenesis. However, these results remain controversial since other studies have not confirmed the link between Q fever and NHL [31][32][33]61].
The aim of the present study was to investigate in vitro the ability of C. burnetii to induce a modulation of the E-cad/β-cat axis during infection. For this purpose, BeWo cells, which are susceptible to C. burnetii infection, were used for their high expression of E-cad at the cell membrane. As a result, we were able to demonstrate that the presence of C. burnetii in the cells triggers overexpression of most of the genes of the signaling pathway, in particular CDH1 genes coding for the E-cad protein. Indeed, E-cad is known as a tumor suppressor trans-membrane protein that inhibits β-cat activity by sequestrating it into the cell cytoplasm through interaction via the E-cad cytoplasmic domain. In addition, cleavage of the E-cad protein by proteases (known as sheddases) and excretion of a soluble fraction of E-cadherin (sE-cad) can contribute to cellular transformation through the loss of cell-to-cell adhesion [62]. This is considered to be a triggering event, promoting translocation of β-cat to the nucleus, where it forms complexes with BCL9 and T-cell factor (TCF), leading to transactivation of cell cycle genes such as cyclin kinase D1/CCND1 and increased cell proliferation [63,64]. Moreover, the generation of sE-cad through sheddases and the subsequent nuclear re-localization of β-cat may be a critical indicator for cancer development. The induction of carcinogenic cascades via the E-cad/β-cat axis was reported in gastric adenocarcinoma associated with Helicobacter pylori infection, in which the bacterium activates calpain sheddase [65], or in myeloid-cell dependent distal colon tumorigenesis associated with Bacteroides fragilis toxin, a known bacterial sheddase [66,67]. We recently reported [30] that E-cad is cleaved in cells infected by C. burnetii. We demonstrated that, in addition to modulating CDH1 expression at the transcriptomic level, infection with C. burnetii leads to disruption of E-cad at the cellular surface of BeWo cells. Under infection conditions, we observed a reduction of E-cad expression at the cell surface that is likely due to cleavage of the extracellular fraction of the protein, as its concentration in the supernatant is continuously increasing over time and the CDH1 gene is overexpressed. In BeWo cells infected with live C. burnetii, we observed a remodeling of the actin filament, which triggers a morphological change. This particular effect was reported for the first time in the study of Meconi et al. [59,68], in which they showed that virulent C. burnetii stimulated morphological changes in human monocytes via the reorganization of the actin cytoskeleton. As E-cad is linked to the actin cytoskeleton through β-cat and α-cat, a rearrangement of the actin bundle can modify the adhesive function of E-cad and play an active role in the migratory activity of carcinoma cells [69]. In the case of C. burnetii infection, the bacterium, through the disruption of E-cad, may exploit the actin cytoskeleton to modulate its internalization by host cells. An interesting observation that emerged from this study concerns the decreased amount of β-cat protein in the cells, even though it is overexpressed at the transcriptomic level in cells infected with the live bacterium. As β-cat is part of the canonical Wnt-Frizzled/ β-cat pathway, we investigated most of the gene operating in the axis and as a result the expression of the genes WNT3A (encoding the extracellular Wnt molecules that bind Frizzled/low-density lipoprotein-receptor related proteins (LPR) complex and act on the canonical Wnt signaling pathway), FZD1/Frizzled (Wnt receptor), NKD2 (Wnt pathway regulator protein) were significantly overexpressed in cells infected with live bacterium, mainly those infected with the more virulent Guiana strain (Cb175). Interestingly, it has been shown that the activation of the Wnt/ Frizzled//β-catenin pathway led to altered expression of genes involved in cell cycle regulation and apoptosis in normal and leukemic B-cell progenitors [70]. In a previous publication, an overexpression of transcription factor TCF7L2 and BCL9 was found, which is known to promote tumor progression by conferring enhanced proliferation, metastatic, and angiogenic properties to cancer cells [71]. This may indicate that in the extremely rare cases C. burnetii infection possibly activates signal which could contribute in part to lymphoma initiation. As we have already pointed out, a recent large analysis of the Dutch population, concluded to the lack of increased risk of NHL after Q fever [33], indicating that infection by C. burnetii alone does not seem to be the cause of an increase in NHL and that other co-factors must be sought. Furthermore, we also note that the expression of the tumor suppressor adenomatous polyposis coli (APC) is repressed in infected cells compared to the other conditions, suggesting that C. burnetii promotes the canonical Wnt signaling pathway. Similar results were obtained from the microarray analysis of the Wnt/Frizzled/β-catenin pathways in BeWo cells gene expression. Obviously, we are well aware of having used in this study a particular cell model, a human trophoblastic cell line, and that under these conditions there is a risk to overstate the biological significance of our data with the aim of explaining certain aspects of the pathophysiology of Q fever. In fact, B cell analysis, which would have been more physiologically relevant, was not possible because the CD20+E-Cad+ cells represent less than 1% of circulating B cells, as we published in a previous article [30]. However, this BeWo model is very useful for studying the link between infection by C. burnetii and modulation of the expression of E-cad and provided a possible molecular basis for explaining the increase in sE-cad concentrations in sera of Q fever patients [30]. Very recently found evidence that the cleavage of E-cad was mediated by a functional HtrA sheddase encoded in the C. burnetii genome [72].
In conclusion, this work provides for the first time evidence of the disruption of E-cad both at the transcriptomic and cell surface levels by C. burnetii, opening a new avenue of research in understanding the pathophysiology of Q fever. We hypothesize that the cleavage of E-cad by C. burnetii could be a means to ensure its transmigration in order to modulate the reactivity of immune cells and thus facilitate C. burnetii progression towards the target organs.